Molecular contamination of an animal facility during and after African swine fever virus infection.

Introduction: The molecular contamination of an animal facility was investigated during and after an infection with highly pathogenic African swine fever virus (ASFV) among domestic pigs. The investigation evaluated the risk of indirect transmission of the disease and indicated points that may facilitate cleaning and disinfection processes. Material and Methods: Six domestic pigs were infected oronasally with the highly pathogenic Georgia 2007 strain. Environmental samples from the floors, walls, rubber floor mats, feeders, drinkers, high-efficiency particulate-absorbing filter covers and doors were collected 7 days post infection (dpi), 7 days later and 24 h after disinfection of the facility. The samples were investigated by real-time PCR and in vitro assays to find genetic traces of ASFV and infectious virus. Results: Typical clinical outcomes for ASF (i.e. fever, apathy, recumbency and bloody diarrhoea) were observed, and all animals died or required euthanasia before or at 9 dpi. No infectious virus was found in environmental samples at the sampling time points. Genetic traces of ASFV were found in all locations except the doors. The initial virus load was calculated using real-time PCR threshold cycle values and was the highest at the drain. A statistically significant decrease of virus load over time was found on non-porous surfaces mechanically cleaned by water (the floor and drain). Conclusion: The gathered data confirmed different routes of virus excretion (oral and nasal, faeces and urine, and aerosol) and showed virus locations and different initial concentrations in the animal facility. Maintaining the facility with mechanical cleaning and using personal protection (gloves) and hand disinfection may efficiently minimise the risk of further virus spread. Together with the results of previously published studies, the present investigations' failure to isolate infectious virus may suggest that if stable environmental conditions are assured, the time needed before the introduction of new herds into previously ASF-affected farm facilities could be shortened and in this way the economic losses caused by the disease outbreak mitigated.

Feeding Spray-Dried Porcine Plasma to Pigs Improves the Protection Afforded by the African Swine Fever Virus (ASFV) BA71∆CD2 Vaccine Prototype against Experimental Challenge with the Pandemic ASFV-Study 2.

This study aimed to evaluate the effects of feeding spray-dried porcine plasma (SDPP) on the protection afforded by the BA71∆CD2 African swine fever virus (ASFV) vaccine prototype. Two groups of pigs acclimated to diets without or with 8% SDPP were intranasally inoculated with 105 plaque-forming units (PFU) of live attenuated ASFV strain BA71∆CD2 and, three weeks later, left in direct contact with pigs infected with the pandemic Georgia 2007/01 ASFV strain. During the post-exposure (pe) period, 2/6 from the conventional diet group showed a transient peak rectal temperature >40.5 °C before day 20 pe, and some tissue samples collected at 20 d pe from 5/6 were PCR+ for ASFV, albeit showing Ct values much higher than Trojan pigs. Interestingly, the SDPP group did not show fever, neither PCR+ in blood nor rectal swab at any time pe, and none of the postmortem collected tissue samples were PCR+ for ASFV. Differential serum cytokine profiles among groups at vaccination, and a higher number of ASFV-specific IFNϒ-secreting T cells in pigs fed with SDPP soon after the Georgia 2007/01 encounter, confirmed the relevance of Th1-like responses in ASF protection. We believe that our result shows that nutritional interventions might contribute to improving future ASF vaccination strategies.

Cross-protection against African swine fever virus upon intranasal vaccination is associated with an adaptive-innate immune crosstalk.

African swine fever virus (ASFV) is causing a worldwide pandemic affecting the porcine industry and leading to important global economic consequences. The virus causes a highly lethal hemorrhagic disease in wild boars and domestic pigs. Lack of effective vaccines hampers the control of virus spread, thus increasing the pressure on the scientific community for urgent solutions. However, knowledge on the immune components associated with protection is very limited. Here we characterized the in vitro recall response induced by immune cells from pigs intranasally vaccinated with the BA71ΔCD2 deletion mutant virus. Vaccination conferred dose-dependent cross-protection associated with both ASFV-specific antibodies and IFNγ-secreting cells. Importantly, bulk and single-cell transcriptomics of blood and lymph node cells from vaccinated pigs revealed a positive feedback from adaptive to innate immunity. Indeed, activation of Th1 and cytotoxic T cells was concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages, as well as CXCL10-expressing lymphocytes and cross-presenting dendritic cells. Altogether, this study provides a detailed phenotypic characterization of the immune cell subsets involved in cross-protection against ASFV, and highlights key functional immune mechanisms to be considered for the development of an effective ASF vaccine.

Higher chronic stress and less satisfaction with GP communication in migrants with unemployment experience: data from the representative German DEGS1 and the GPCare-1 study.

BACKGROUND: The impact of unemployment on health is well studied. However, information on associations of unemployment, migration background and general practitioner-patient communication is scarce. METHODS: Data from the representative German Health Interview and Examination Survey for Adults (DEGS1) of individuals in working age (n = 5938) were analysed stratified by unemployment and migration background. Using official weighting factors, the prevalence of chronic stress, having ≥1 chronic disease, having a GP and GP visits in the last 12 months was determined. Multivariate regression models were analysed for associations between unemployment, migration background, and other socio-demographic characteristics with GP visits and chronic stress. Data from the General Practice Care-1 (GPCare-1) study (n = 813 patients) were analysed for differences in patient-physician communication between unemployed with and without migration background. Reverse proportional odds models were estimated for associations of unemployment and migration background with physician-patient communication. RESULTS: In the DEGS1, 21.5% had experienced unemployment (n = 1170). Of these, 31.6% had a migration background (n = 248). Compared to unemployed natives, unemployed with migration background had higher chronic stress (mean: 14.32 vs. 13.13, p = 0.02), while the prevalence of chronic disease was lower (21.7% vs. 30.2%, p = 0.03). They were less likely to have a GP (83.6% vs. 90%, p = 0.02), while GP visits were similar (mean: 3.7 vs. 3.3, p = 0.26). Migration background and unemployment experience were not associated with GP visits, while both factors were significantly associated with higher chronic stress (both: p < 0.01). In GPCare-1, 28.8% had ever experienced unemployment (n = 215). Of these, 60 had a migration background (28.6%). The unemployed with migration background reported less frequently that the GP gives them enough space to describe personal strains (46.5% vs. 58.2%; p = 0.03), and that their problems are taken very seriously by their GP (50.8% vs. 73.8%; p = 0.04). In multivariate analyses, migration background showed a lower probability of having enough space to describe personal strains and feeling that problems were taken very seriously. CONCLUSION: Unemployment experience and migration background were associated with higher chronic stress. Only migration background was associated with less satisfaction regarding physician-patient communication.

Development of an inactivated combined vaccine for protection of cattle against lumpy skin disease and bluetongue viruses.

Lumpy Skin Disease (LSD) and Bluetongue (BT) are the main ruminants viral vector-borne diseases. LSD is endemic in Africa and has recently emerged in Europe and central Asia as a major threat to cattle industry. BT caused great economic damage in Europe during the last decade with a continuous spread to other countries. To control these diseases, vaccination is the only economically viable tool. For LSD, only live-attenuated vaccines (LAVs) are commercially available, whilst for BT both LAVs and inactivated vaccines are available with a limited number of serotypes. In this study, we developed an inactivated, oil adjuvanted bivalent vaccine against both diseases based on LSDV Neethling strain and BTV4. The vaccine was tested for safety and immunogenicity on cattle during a one-year period. Post-vaccination monitoring was carried out by VNT and ELISA. The vaccine was completely safe and elicited high neutralizing antibodies starting from the first week following the second injection up to one year. Furthermore, a significant correlation (R = 0.9040) was observed when comparing VNT and competitive ELISA in BTV4 serological response. Following BTV4 challenge, none of vaccinated and unvaccinated cattle were registered clinical signs, however vaccinated cattle showed full protection from viraemia. In summary, this study highlights the effectiveness of this combined vaccine as a promising solution for both LSD and BT control. It also puts an emphasis on the need for the development of other multivalent inactivated vaccines, which could be greatly beneficial for improving vaccination coverage in endemic countries and prophylaxis of vector-borne diseases.

More GP contacts and poorer health of informal caregivers with low socioeconomic status in Germany: results from the population-based DEGS1 and the cross-sectional GPCare-1 study.

OBJECTIVES: Informal caregivers are known to have poorer mental health. Risk factors for caregiver burden include low education, female gender, cohabitation with the care recipient and lack of resources. General practitioners (GPs) have an important role in supporting caregivers. Drawing on data from two surveys, associations between caregivers' socioeconomic status (SES), psychophysical health and GP contacts are analysed. DESIGN: Cross-sectional study. The study draws on data from two surveys (German Health Interview and Examination Survey for Adults, DEGS1 and General Practice Care-1, GPCare-1). SETTING: Germany. PARTICIPANTS: DEGS1: German general population (18+ years) n=7987. GPCare-1: general practice patients (18+ years) n=813. PRIMARY OUTCOME: Psychophysical health, GP contacts and communication. METHODS: Using representative DEGS1 data, the prevalence of informal caregivers, caregivers' burden, chronic stress, various health conditions and frequency of GP contacts were evaluated stratified by SES. Data from the GPCare-1 study addressed caregivers' experiences and communication preferences with GPs. RESULTS: In the DEGS1, the prevalence of caregivers was 6.5%. Compared with non-caregivers, caregivers scored significantly higher for chronic stress (15.45 vs 11.90), self-reported poor health (37.6% vs 23.7%) and GP visits last year (3.95 vs 3.11), while lifestyle and chronic diseases were similar. Compared with caregivers with medium/high SES, those with low SES had a significantly lower prevalence of high/medium caregiver burden (47.9% vs 67.7%) but poorer self-reported health (56.9% vs 33.0%), while other characteristics did not differ. In the GPCare-1 study, the prevalence of caregivers was 12.6%. The majority of them felt that their GP takes their problems seriously (63.6%) without difference by SES. CONCLUSION: Caregivers with low SES constitute an especially high-risk group for psychological strain, requiring special GP attention to support their needs.

Development and Evaluation of an Inactivated Lumpy Skin Disease Vaccine for Cattle.

Lumpy skin disease (LSD) of cattle is caused by a virus within Capripoxvirus genus. It leads to huge economic losses in addition to trade and animal movement limitation. Vaccination is the only economically feasible way to control this vector-borne disease. Only live attenuated vaccines have been used so far and no inactivated vaccine has been developed nor tested in cattle. In this study, we developed an inactivated oily adjuvanted vaccine based on Neethling strain and tested it on cattle. Selected criteria of appreciation were safety, antibody response by Virus Neutralization and protection through challenge. A field trial was also performed in Bulgaria. The vaccine was safe and did not cause any adverse reaction, high level of specific antibodies was obtained starting from day 7 post-vaccination and protection against virulent challenge strain that caused typical disease in control animals was total. Induced protection was similar to that obtained with live vaccine, without any adverse effect. In addition, the field study confirmed safety and efficacy of the vaccine, which did not show any adverse reaction and induced a high level of antibodies for up to one year. General prophylaxis based on inactivated vaccine could be of great benefit in endemic countries or at risk regions.

Structural characterization of the PCV2d virus-like particle at 3.3 Šresolution reveals differences to PCV2a and PCV2b capsids, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes.

Porcine circovirus 2 (PCV2) has a major impact on the swine industry. Eight PCV2 genotypes (a-h) have been identified using capsid sequence analysis. PCV2d has been designated as the emerging genotype. The cryo-electron microscopy molecular envelope of PCV2d virus-like particles identifies differences between PCV2a, b and d genotypes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. These differences indicate that sequence analysis of genotypes is insufficient, and that it is important to determine the PCV2 capsid structure as the virus evolves. Structure-based sequence comparison demonstrate that each genotype possesses a unique combination of amino acids located on the surface of the capsid that undergo substitution. We also demonstrate that the capsid N-terminus moves in response to increasing amount of nucleic acid packaged into the capsid. Furthermore, we model a tetranucleotide between the 5- and 2-fold axes of symmetry that appears to be responsible for capsid stability.

Recognition of heparan sulfate by clinical strains of dengue virus serotype 1 using recombinant subviral particles.

Dengue is the most important arthropod-borne viral disease in humans, with an estimated 3.6 billion people at risk for infection and more than 200 million infections per year. Identification of the cellular receptors for dengue virus (DV), the causative agent of dengue, is important toward understanding the pathogenesis of the disease. Heparan sulfate (HS) has been characterized as a DV receptor in multiple model systems, however the physiological relevance of these findings has been questioned by observations that flaviviruses, including DV, can undergo cell culture adaptation changes resulting in increased binding to HS. It thus remains unclear whether HS is utilized by clinical, non-cell culture-adapted strains of DV. To address this question, herein we describe a set of methodologies using recombinant subviral particles (RSPs) to determine the utilization of HS by clinical strains of DV serotype 1 (DV1). RSPs of clinically isolated strains with low cell culture passage histories were used to study HS interaction. Biochemically characterized RSPs showed dose-dependent binding to immobilized heparin, which could be competed by heparin and HS but not structurally related glycosaminoglycans chondroitin sulfate A and hyaluronic acid. The relevance of heparin and HS biochemical interactions was demonstrated by competition of RSP and DV binding to cells with soluble heparin and HS. Our results demonstrate that clinical strains of DV1 can specifically interact with heparin and HS. Together, these data support the possibility that HS on cell surfaces is utilized in the DV-human infection process.

Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins.

Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of baculovirus-expressed E(rns), E1, and E2 proteins on immunogenicity. Interestingly, E(rns), E1, and E2 proteins lacking proper post-translational modifications, most noticeable lack of glycosylation, failed to induce a detectable virus neutralizing antibody (NA) response and protection against CSFV. Similarly, no NA or protection was observed in pigs immunized with E1 glycoprotein. Analysis of E(rns) and E2 proteins with single site glycosylation mutations revealed that detectable antibody responses, but not protection against lethal CSFV challenge is affected by removal of specific glycosylation sites. In addition, it was observed that single administration of purified E(rns) glycoprotein induced an effective protection against CSFV infection.

Identification of an NTPase motif in classical swine fever virus NS4B protein.

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.

Human herpesvirus-6 in patients with Crohn's disease.

Human herpesvirus-6 (HHV-6) infections are usually asymptomatic reactivations in immunocompetent persons, but may be severe in immunocompromised individuals. Although primary HHV-6 infection is mainly associated with roseola infantum, it has also been associated with gastroenteritis, diarrhea, and nausea in children. In this study, we investigated the potential role of HHV-6 in Crohn's disease (CD). Evidence of HHV-6 infection in CD patients and controls was determined by immunohistochemistry (IHC), polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR). Fifty-one tissue blocks from 23 CD patients and 20 tissue blocks from 20 controls were examined. Quantitativereal-time PCR was used to assess HHV-6 viral loads. IHC, PCR and qPCR indicated the presence of HHV-6 in both CD patients and controls. Immunohistochemistry of tissues revealed an almost equal frequency and distribution of positive cells; however, non-specific immunostaining confounded interpretation. HHV-6 DNA was detected in 52% (12/23) of CD and 55% (11/20) of control patients by PCR and in 69.5% (16/23) of CD cases and 65% (13/20) of controls by qPCR. Mean viral load in intestinal tissues was similar in CD and controls (33.4 and 57.9 copies microg(-1) DNA, respectively). Finding equal evidence of HHV-6 in patients and controls by multiple methods suggests that this virus is ubiquitous and probably not a cause of CD.

Diversified reassortant H9N2 avian influenza viruses in chicken flocks in northern and eastern China.

According to our previous study of the M genes of H9N2 avian influenza viruses (AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly emerged in northern and eastern China in addition to the existing A/chicken/Hong Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of seventeen H9N2 isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.

Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ.

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.

Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.